1. Field of the Invention
The present invention relates to decellularized grafts for tissue engineering, and more particularly but not by way of limitation, to decellularized grafts from umbilical cord vessels for tissue engineering as an acellular matrix and for use with cell seeding methodology.
2. Brief Description of the Art
Vascular disease is the number one cause of death in Western societies. Current treatments are limited to reconstructive surgery where a patient's own (autologous) non-diseased vessels are relocated to bypass diseased or blocked blood vessels that would ultimately result in cardiac arrest, stroke or death.
Maintaining the flow of oxygen-rich blood to organs down-stream of severely occluded blood vessels is often limited to surgically by-passing diseased sections with substitute vessels. Transplanted autologous arteries are considered the “gold standard” for small diameter replacement vessels (<6 mm), with patency rates of approximately 90% at five years. By comparison, patency rates of 25-45% over 5 years are typical with synthetic alternatives such as Dacron or expanded polytetrafluoroethylene (ePTFE). Approximately 30% of all patients requiring vascular reconstructions do not have ‘autologus’ vessels available for use, necessitating the use of either a synthetic conduit or a non-operative approach. Small diameter (<6 mm) vascular reconstructions have proven considerably more problematic than large diameter vessel reconstructions, where reduced diameter and low flow rates, compounded by high resistance, amplify poor biological host/graft interactions. This inability to interact successfully with recipient tissues initiates a complex set of adverse biological reactions that can be broadly grouped into thrombotic and hyperplastic responses. Autologous arterial grafts are preferred over synthetics because the latter fail due to poor biocompatibility and/or mechanical properties.
Alternative approaches to improve the current situation include the development of synthetic and biological grafts, endothelial cell seeded synthetic grafts and tissue engineering. Processed biological materials have been used clinically as vessel replacements; these include cryo-preserved and cross-linked vessels such as the saphenous and umbilical veins. A distinct and important advantage of processed biological tissues over current synthetic materials is the retention of native-like mechanical properties, where the compliance (or mechanical) matching is predictive of graft success (Seifalian et al., 1999). Further, the physical and chemical environment within these materials is inherently more conducive to native vessel remodelling processes.
The umbilical cord vein has an established record as an inert ‘fixed’ biomaterial. However, there are several structural limitations when the umbilical cord vein is used as a glutaraldehyde ‘fixed’ material. Although cryo-preserved veins are a promising option, they too are limited by supply (Dresdale et al., 1990; Fujitani et al, 1992; and Davies, 1994), and umbilical cord veins, in their current form, have not proven reliable due (primarily) to a potential for aneurysm formation. With an incidence of 33% beginning at three years and increasing with time, the HUV has not seen wide spread usage (Hasson et al., 1986; Nevelsteen et al., 1988; and Karkow et al., 1986). However, other more favorable data suggests the HUV does in actual fact out perform PTFE, with patency rates 2.1 times higher (Eickhoff et al., 1987). Dardik et al. (1982), who developed the glutaraldehyde tanned HUV graft (Dardik I et al., 1973), also reported aneurysm formation, but has remarked that due to the poor patency rates of a PTFE graft the HUV is still an “acceptable alternative to the absent or deficient autologous vein”. More recently Johnson et al. (2000), have reiterated the suitability of biological grafts over synthetic alternatives, with results from a five year primary patency study of 80% for saphenous vein (SV), 56% HUV and 33% for PTFE (Johnson et al., 2000).
The use of collagen based biomaterials has traditionally necessitated a degree of tissue processing to stabilize and prevent chronic immune responses against foreign epitopes (Khor, 1997; and Schmidt et al., 2000). These treatments have been applied to the umbilical vein graft to improve its biocompatibility and mechanical strength under arterial conditions (Karkow et al., 1986; Dardik H et al., 1984; Dardik, 1990; Dardik, 1995; and Miyata et al., 1989). The mechanism of stabilization is through covalent cross-linking of the collagen fibers within the ECM, rendering the material resistant to host enzymatic degradation. The reduced immune response attributed to cross-linking has been described as a ‘masking’ of allogenic or xenogenic components that would otherwise be seen as foreign, resulting in chronic rejection and subsequent failure of the graft. The quandary is that chemical treatments designed to stabilize and reduce immunogenicity, often inherently cytotoxic, prevent cell migration, and as such, true functionality of the graft will never be achieved. A number of acellular collagen based matrices (not cross-linked) have been studied: vascular (Teebken et al., 2000; Courtman et al., 2000; Badylak et al., 1998; and Badylak et al., 1999), bladder augmentation (Probst et al., 2000; and Probst et al., 1997) and cardiac valves (Courtman et al., 1995; Courtman et al., 1994; and Bader et al., 1998). These are promising studies with cells migrating into and populating the matrix material, showing that cross-linking is not necessarily a vital step. However, Courtman et al. (2000) found that, despite decellularization, immunogenic proteins remained localized within the vascular graft media (not the graft periphery), concluding that immunogenic proteins “arise from proteins associated with the distinct extracellular arterial immunogenic matrix”. Although these materials are promising, problems of thrombosis, neointimal hyperplasia and graft degradation have meant that translating these results into viable grafts has proven a significant hurdle.
In order to replicate the success of autologous arterial transplants, a successful prosthetic must integrate and function in a similar manner to natural arteries. It is the failure of current small diameter prosthetics to integrate appropriately with recipient tissue that initiates a number of unfavorable biologic interactions cumulating in thrombotic and hyperplastic responses that lead to graft failure (Schmidt et al, 1999). To improve the host/graft interaction, it is likely that both a competent endothelium, to serve at the blood-graft interface, and a fully developed, biocompatible vascular wall, populated with vascular smooth muscle cells (VSMC), must be present. The logic behind this approach is clear: grafts frequently fail due to poor functional integration, and therefore to improve function a biologic component must be present. It follows that if the biologic component is more comprehensive, then it is likely improved biologic function will result. As neither a functional vessel wall nor an endothelium will spontaneously develop in adult humans (at an appreciable rate), tissue engineering offers a unique methodology where replacement neo-vessels can be grown in vitro (Nikalson et al., 1999; Nerem et al., 2001; Langer et al., 2000; and L'Heureux et al., 1998). By incorporating functional cell lineages into 3D scaffolds, or blood vessel templates, improved biologic function can be achieved to minimize intrinsic host repair or defense mechanisms that would otherwise lead to the aforementioned thrombotic and hyperplastic responses.
A key component of this process is the choice of 3D scaffold with which tissue growth is guided. The list of 3D scaffold materials continues to grow, and includes the following: permanent synthetics (Deutsch et al., 1999), biodegradable synthetics (Hoerstrup et al., 2001; and Niklason et al., 1997), or variously treated ex vivo materials from either human or animal origin (Khor et al., 1997; Niklason et al., 1999; McFetridge et al., 2004; Schaner et al., 2004, and Hiles et al., 1995). The ideal vascular scaffold is required to be biocompatible and ideally have in vivo-like mechanical properties with the capacity to guide, support, and maintain cellular function. Compared to many synthetic polymers, processed ex vivo materials often lack mechanical uniformity, consistency, composition, and can be restrictive in their final shape/structure. Extraction of foreign epitopes to reduce the immunogenicity of ex vivo materials is clearly an important issue. Methods of tissue processing that extract immunogenic components have been shown to be relatively successful at reducing the immune impact of these ex vivo biomaterials (Schaner et al., 2004; and Hiles et al., 1995). The clinical use of collagen hydrogels in cosmetic surgery and the small intestinal submucosa (SIS) have validated the use of these materials (Chen et al., 2001; Hiles et al., 1995; and Lantz et al., 1993). Although ex vivo tissue processing is an effective means to reduce the immunogenic load, mass transfer limitations of thicker/larger organs are likely to reduce processing efficiency. A distinct and important advantage of ex vivo vascular derived scaffolds is that the physical and chemical environment is inherently more conducive to cell adhesion and native remodeling processes than many synthetic alternatives. For example, cell adhesion is enhanced due to endogenous RGD adhesion sequences present within the amino acid sequence of extracellular matrix (ECM) collagen (Saito et al. 2001), and the retention of the native blood vessels' mechanical properties (compliance matching) is an important predictor of graft success (Tai et al., 2000; Roeder et al., 1999; and Seifalian et al., 1999).
The human umbilical vein (HUV) has a comprehensive clinical history as a glutaraldehyde fixed bypass graft (Dardik et al., 1973; Dardik et al., 1976; Dardik et al., 1988; Dardik et al., 1976; Dardik et al., 1995; and Dardik, 2001). However, time consuming and error prone manual dissection procedures result in a lack of mechanical uniformity, limiting the use of this material as a ‘stand-alone scaffold’ (without additional support), as a biomaterial for tissue engineering applications. The HUV has a number of properties that show promise as an acellular 3D vascular scaffold: (1) it has the structure and form of a natural blood vessel to more closely replicate arterial compliance; (2) its allograft origin reduces the risk of interspecies viral contamination; and (3) because of its vascular derivation, it presents surfaces that are conducive to cellular attachment and subsequent remodeling processes (McFetridge et al., 2004; McFetridge, 2002; Teebken, 2004; and Teebken et al., 2000). With lengths that can exceed 500 mm and internal diameters from 4-6 mm, the HUV is appropriate for several vascular reconstructive applications.
Therefore, there exists a need in the art for new and improved biomaterials derived from ex vivo tissues that offer a viable alternative for use as tissue engineering scaffolds. It is to such a decellularized graft from an umbilical cord vessel, as well as methods of preparing and using same, that the present invention is directed.